Dickerson J B, Blackwell J E, Ou J J, Shinde Patil V R, Goetz D J
Department of Biomedical Engineering, University of Memphis, Memphis, Tennessee, USA.
Biotechnol Bioeng. 2001 Jun 20;73(6):500-9. doi: 10.1002/bit.1085.
In a variety of disease settings the expression of the endothelial selectins E- and P-selectin appears to be increased. This feature makes these molecules attractive targets around which to design directed drug-delivery schemes. One possible approach for achieving such delivery is to use polymeric biodegradable microspheres bearing a humanized monoclonal antibody (MAb) for E- and P-selectin, MAb HuEP5C7.g2. Perhaps the simplest technique for "coupling" HuEP5C7.g2 to the microspheres is via nonspecific adsorption. Previous studies suggest, however, that the adsorption of proteins onto microspheres fabricated in the presence of a stabilizer such as poly(vinyl alcohol) (PVA) is limited. It is unclear to what extent this limited level of adsorbed HuEP5C7.g2 would be able to support adhesion to E- and P-selectin under flow conditions. To explore this issue, we prepared microspheres from the biodegradable polymer, poly(epsilon-caprolactone) (PCL), using a single emulsion process and PVA as a stabilizer. We then incubated the PCL microspheres with HuEP5C7.g2 and studied the adhesion of the resulting HuEP5C7.g2 microspheres to E- and P-selectin under in vitro flow conditions. We found that the HuEP5C7.g2 PCL microspheres exhibit specific adhesion to Chinese hamster ovary cells stably expressing P-selectin (CHO-P) and 4-h IL-1beta-activated human umbilical vein endothelial cells (HUVEC). In contrast, HuEP5C7.g2 PCL microspheres exhibit little adhesion to parental CHO cells or unactivated HUVEC. The attachment efficiency to the selectin substrates was quite low, with appreciable attachment occurring only at low shear (0.3 dyn/cm(2)). Other supporting data strongly suggest that the limited attachment efficiency is due to a low level of HuEP5C7.g2 adsorbed to the PCL microspheres. Although the attachment was limited, a significant percentage of the HuEP5C7.g2 PCL microspheres were able to remain adherent at relatively high shear (8 dyn/cm(2)). Combined, our data suggest that HuEP5C7.g2 PCL microspheres exhibit selective limited adhesion to cellular substrate expressing E- and P-selectin.
在多种疾病情况下，内皮细胞选择素E选择素和P选择素的表达似乎会增加。这一特性使得这些分子成为设计定向药物递送方案的有吸引力的靶点。实现这种递送的一种可能方法是使用带有针对E选择素和P选择素的人源化单克隆抗体（MAb）HuEP5C7.g2的可生物降解聚合物微球。也许将HuEP5C7.g2“偶联”到微球上最简单的技术是通过非特异性吸附。然而，先前的研究表明，在诸如聚乙烯醇（PVA）等稳定剂存在下制备的微球上蛋白质的吸附是有限的。目前尚不清楚这种有限水平的吸附HuEP5C7.g2在流动条件下能够在多大程度上支持与E选择素和P选择素的粘附。为了探讨这个问题，我们使用单乳液法和PVA作为稳定剂，从可生物降解聚合物聚（ε-己内酯）（PCL）制备了微球。然后我们将PCL微球与HuEP5C7.g2一起孵育，并在体外流动条件下研究所得HuEP5C7.g2微球与E选择素和P选择素的粘附。我们发现HuEP5C7.g2 PCL微球对稳定表达P选择素的中国仓鼠卵巢细胞（CHO-P）和经4小时白细胞介素-1β激活的人脐静脉内皮细胞（HUVEC）表现出特异性粘附。相比之下，HuEP5C7.g2 PCL微球对亲本CHO细胞或未激活的HUVEC几乎没有粘附。对选择素底物的附着效率相当低，仅在低剪切力（0.3达因/平方厘米）下才会有明显的附着。其他支持数据有力地表明，有限的附着效率是由于吸附到PCL微球上的HuEP5C7.g2水平较低。尽管附着是有限的，但相当大比例的HuEP5C7.g2 PCL微球能够在相对较高的剪切力（8达因/平方厘米）下保持粘附。综合来看，我们的数据表明HuEP5C7.g2 PCL微球对表达E选择素和P选择素的细胞底物表现出选择性有限粘附。
